tbuckley/cabot-search huggingface dataset:
Abstract 907: Evaluation of the anti-GPC1 scFv-Fc fusion protein as a detection tool of GPC1 expression in human cancers
Authors: Takeshi Sawada, Tomonori Yaguchi, Yuko Sakai, Chinatsu Suzuki, Kenji Morii, Masahiko Aoki, Masaru Fukahori, Satoshi Serada, Tetsuji Naka, Jun K. Yamashita, Yutaka Kawakami, Takako E. Nakajima
Journal: Cancer Research (2023)

Abstract Background: We have developed a glypican-1 (GPC1) targeted CAR-T cell with strong anti-tumor effects against solid tumors in vivo (Kato et al 2020 eLife). In general, an antibody is used for detection of target protein expression in the tumor to identify appropriate targets for the CAR-T cell therapy in solid tumors. However, the local conjunction between CAR-T cells and the target is not exactly the same in the antibody and the target because CAR-T cell recognizes the target by single-chain variable fragment (scFv). In this study, we evaluated the anti-GPC1 scFv-Fc fusion protein as a detection tool of GPC1 expression in human cancer cell lines. Methods: The sequences encoding the anti-GPC1 scFv are based on the sequence of anti-GPC1 mAb (clone: 1-12) that recognizes human and mouse GPC1 (Harada et al., 2017). The sequences of anti GPC-1 scFv were cloned into pCAG-Neo mIgG2a-Fc plasmid for the generation of anti GPC1_scFv-mIgG2a_Fc fusion proteins. The plasmids were transfected into 293 T cells and the fusion proteins were purified from their culture supernatants using Antibody TCS Purification Kit. LK2-GPC1 (a GPC1 overexpressing lung carcinoma cell line), TE8 (an esophageal carcinoma cell line with endogenous GPC1) and LK2-mock (a GPC1-negative lung carcinoma cell line) were used for the evaluation of the anti-GPC1 scFv-Fc fusion protein. Results: In FACS assays, fluorescence microscopy analysis and immunohistochemistry staining, GPC1 expression was successfully detected by both anti-GPC1 scFv of VL-VH and VH-VL in LK2-GPC1 and TE-8, and not detected in LK2-mock. Localization of GPC1 expression on the cell membrane was confirmed by fluorescence microscopy. Perspective: Further comparative evaluation between anti-GPC1 antibody and anti-GPC1 scFv-Fc fusion proteins are needed especially in human tumor tissues. We plan to evaluate a relationship between GPC1 positivity in various human tumor tissues detected by anti-GPC1 scFv-Fc fusion protein and CAR-T cell cytotoxicity against the tissue. Citation Format: Takeshi Sawada, Tomonori Yaguchi, Yuko Sakai, Chinatsu Suzuki, Kenji Morii, Masahiko Aoki, Masaru Fukahori, Satoshi Serada, Tetsuji Naka, Jun K. Yamashita, Yutaka Kawakami, Takako E. Nakajima. Evaluation of the anti-GPC1 scFv-Fc fusion protein as a detection tool of GPC1 expression in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 907.
Abstract 871: Accurate estimation of open chromatin and intrinsic biases in bulk and single-cell CUT&amp;Tag data
Authors: Sheng'en Hu, Qingying Chen, Chongzhi Zang
Journal: Cancer Research (2023)

Abstract Histone modifications (HM) and transcription factors (TF) are aberrantly regulated in the pathogenesis of humans and are a major class of cancer cell dependencies. Precise detection of HM and TF binding genome-wide is essential for a better understanding of cancer epigenetics. Cleavage Under Targets &amp; Tagmentation (CUT&amp;Tag) is an easy and low-cost epigenomic profiling method that can be performed on a low number of cells and on the single-cell level. A large number of CUT&amp;Tag datasets have been generated in cancer samples, providing a valuable resource. Unbiased analysis of CUT&amp;Tag data is important for cancer epigenetics research. CUT&amp;Tag experiments rely on the hyperactive transposase Tn5 for tagmentation. We performed integrative analysis of CUT&amp;Tag and other high-throughput data, and found that Tn5 is subject to intrinsic sequence insertion biases (intrinsic biases). Preference of Tn5 captured reads toward chromatin accessibility regions (open chromatin biases) also confound the distribution of CUT&amp;Tag reads. Both sources of biases confound the analysis of CUT&amp;Tag data, especially for factors that are not expected to associate with accessible chromatin. High sparsity of single-cell data makes the effect of both biases more substantial compared to bulk data. We present a computational model for accurate characterization and quantitative estimation of biases in CUT&amp;Tag data. This model paved the way for further development of computational tools for improving both bulk and single-cell CUT&amp;Tag data analysis. Citation Format: Sheng'en Hu, Qingying Chen, Chongzhi Zang. Accurate estimation of open chromatin and intrinsic biases in bulk and single-cell CUT&amp;Tag data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 871.
Abstract 822: Target-specific nanoparticle-based strategy for improved therapeutic efficacy to treat pancreatic cancer
Authors: Juan Luis Vivero-Escoto, Mubin Tarannum, Pinku Mukherjee
Journal: Cancer Research (2023)

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for over 90% of the pancreatic cancer cases and is the fourth-leading cause of cancer-related mortality. Unfortunately, despite the tremendous scientific efforts, it is shocking to note that PDAC has the worst prognosis with 5-year survival rate of 10% which has not significantly improved from the past 40 years. The high mortality and awful prognosis of PDAC can be attributed to multifactorial reasons like its intrinsic and acquired resistant behavior, early micrometastatic dissemination, desmoplastic effect and heterogeneities in tumors, rendering the current treatments rather ineffective. Developing reliable methods for the effective treatment of PDAC will have a major impact on the clinical outcome for this deadly disease. Nanoparticle-based drug delivery using mesoporous silica nanoparticles (MSNs) can be a potential alternative for the treatment of PDAC owing to their outstanding properties such as high surface area, tunable pore diameters, external and internal surface, which make this platform suitable for high drug loading and functionalization. We have designed, synthesized and characterized a target-specific, stimuli-responsive MSN platform for the controlled delivery of cisplatin (cisPt) and gemcitabine (Gem) (TAB004-Gem-cisPt-MSNs) with an optimal drug ratio. Our results demonstrate that this delivery system effectively targets tumor associated MUC1 (tMUC1), increases therapeutic efficiency with reduced toxicity in both a syngeneic and a spontaneous mouse model that overexpressed tMUC1. Moreover, the same MSN platform has been used in a sequential treatment where MSNs containing a Sonic Hedgehog (SHh) inhibitor, cyclopamine (CyP), and the Gem-cisPt-MSNs as the main delivery system led to reduction in the tumor stroma along with an improvement in the treatment of PDAC. Taken together, our findings support the potential of drug delivery using MSN nanoparticles for stroma modulation and to prevent pancreatic cancer progression. Citation Format: Juan Luis Vivero-Escoto, Mubin Tarannum, Pinku Mukherjee. Target-specific nanoparticle-based strategy for improved therapeutic efficacy to treat pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 822.
Abstract 898: Generation and validation of anti-linker monoclonal antibodies for the detection of surface expressed scFv-based CARs
Authors: Amrik Singh, Sarah L'Heureux, Ryan Sinapius, Michael Kvorjak, Emma Wade, Jason Lohmueller, Jeremy Fisher, Reginaldo Prioli
Journal: Cancer Research (2023)

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of cells with greater persistence, there is a need in multiple phases of the CAR-T development pipeline for highly specific detection reagents that can be leveraged to monitor the expression of CARs on the cell surface. Many commercially available CAR detection reagents, however, either lack specificity or are not versatile in their ability to detect CARs of differing antigen specificity. Here, we report on the generation and validation of rabbit monoclonal antibodies raised against two linker sequences that are commonly integrated into single-chain variable fragment (scFv)-based CARs. These antibodies serve as versatile detection reagents that can be used to interrogate the surface expression of CARs. Methods: The monoclonal antibodies, E7O2V and E3U7Q, were generated by rabbits immunized with peptide sequences most commonly used to construct the linker region of scFv based CARs, Gly4Ser and Whitlow, respectively. E7O2V and E3U7Q were validated for specificity and versatility using flow cytometric analysis of non-transduced versus CAR-transduced cell lines and primary human T cells. Results: Flow cytometric analysis of live Jurkat cells and primary human T cells transduced with CAR constructs revealed that E7O2V and E3U7Q could detect surface expressed CARs containing the appropriate linker sequence, independently of scFv specificity. No specific staining was observed on non-transduced cells. Conclusions: In a flow cytometry assay, E7O2V and E3U7Q specifically detect surface expressed scFv-based CARs containing either a Gly4Ser linker or a Whitlow linker, respectively. Furthermore, these monoclonal antibodies are versatile in that they can also detect their respective linker sequence independently of scFv specificity. The potential exists to leverage these antibodies for CAR-T cell enrichment and for incorporation into multiparametric flow cytometry panels used to phenotype CAR-T cells during the discovery, manufacturing, and clinical phases of the development pipeline. Citation Format: Amrik Singh, Sarah L'Heureux, Ryan Sinapius, Michael Kvorjak, Emma Wade, Jason Lohmueller, Jeremy Fisher, Reginaldo Prioli. Generation and validation of anti-linker monoclonal antibodies for the detection of surface expressed scFv-based CARs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 898.
Abstract 6049: Exogenous serine promotes cancer stem cells in oral squamous cell carcinoma
Authors: Stacy Ann Jankowski, Nina C. Hardy, Maria A. Kukuruzinska
Journal: Cancer Research (2023)

Abstract Cancer development and progression is associated with metabolic reprogramming of tumor cells required to meet their proliferative, bioenergetic and survival challenges. Some metabolites, including a non-essential amino acid, serine, have been shown to play a role in tumorigenesis by promoting aggressive cell states, such as cancer stem cells (CSCs). Previous studies from our laboratory showed that the nuclear branch of the Wnt/β-catenin signaling pathway, the β-catenin/CBP/MLL1 axis, enhanced CSC states during oral squamous cell carcinoma (OSCC) evolution through H3K4 trimethylation (H3K4me3) and epigenetic remodeling of the chromatin landscape. Given that metabolism has also been shown to modulate cell plasticity via epigenomic modifications, we have aimed to decode the relationship between serine synthesis and cell identity in OSCC. We have shown that under serine starvation conditions, OSCC cells upregulate steady-state mRNA and protein levels of serine synthesis pathway enzymes. The latter is associated with an increase in the by-product, alpha-ketoglutarate (αKG), a co-substrate for nuclear αKG-dependent dioxygenases, which demethylate histone marker H3K27me3 and de-repress differentiation-associated genes. Furthermore, increased production of αKG is associated with downregulation of the β-catenin/CBP/MLl1 complex and CSC-associated H3K4me3. In this study, we used human OSCC cell lines CAL27 and HSC3, derived from a primary tongue tumor and a metastatic site, respectively. Each cell line was cultured in either complete medium or serine starvation medium. Serine starvation conditions led to a downregulation of H3K27me3 along with H3K4me3, promoting a switch from cancer stem cell to differentiation-enhancing chromatin landscape. The observed changes in cell plasticity were further investigated through tumorsphere formation, a surrogate assay for cancer stem cells. We found that OSCC CAL27 and HSC3 cells readily formed tumorspheres replete with stem cell markers BMI1, KRT14 and SOX2, under non-adhesive conditions when grown in complete media, supporting our findings that utilization of exogenous serine enables OSCC cells to maintain stemness. Our findings suggest that a switch from exogenous serine uptake to the endogenous serine biosynthesis promotes OSCC cell differentiation concomitant with the loss of CSC identity. Citation Format: Stacy Ann Jankowski, Nina C. Hardy, Maria A. Kukuruzinska. Exogenous serine promotes cancer stem cells in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6049.
Abstract 6047: ZFTA-RELA fusion aberrantly drives glutamine metabolism in lethal pediatric ependymomas
Authors: Siva Kumar Natarajan, James Haggerty-Skeans, Joanna Lum, Pranav Narayanan, Stefan Sweha, Sushanth Sunil, Pooja Panwalkar, Jill Bayliss, Peter Sajjakulnukit, Derek Dang, Abhinav Achreja, Deepak Nagrath, Costas Lyssiotis, Sriram Venneti
Journal: Cancer Research (2023)

Abstract Ependymomas are fatal brain malignancies with very few treatment options. More than 70% of supratentorial (ST) ependymomas harbor fusions of the zinc-finger containing, chromatin modifier ZFTA and the transcriptional activator of NF-κB signaling, RELA. Oncogene-driven metabolic reprogramming is a fundamental hallmark of cancer that enables sustained tumor proliferation. ZFTA-RELA fusion protein is essential for tumorigenesis and our goal is to determine how it drives metabolism in ST-ependymomas. To address this, we developed an in vitro isogenic system by expressing the ZFTA-RELA fusion protein in immortalized mouse neural stem cells (ZFTA-RELAFUS). Using this system, we show that these tumor cells selectively upregulate expression of the glutamine transporter (SLC1A5), glutaminase (GLS), and many downstream enzymes in the glutamine metabolic pathway. Therefore, we hypothesized that the ZFTA-RELA fusion drives glutamine metabolism in ST-ependymomas. We further demonstrate that ZFTA-RELAFUS tumor cells utilize glutamine to maintain redox homeostasis and show marked cell death upon its withdrawal. Moreover, JHU-083, a specific pharmacologic inhibitor of glutaminase, killed ZFTA-RELAFUS tumor cells in vitro and in vivo. To summarize, our results suggest that the ZFTA-RELA fusion expressing tumor cells exhibit strong glutamine dependence, and targeting it has significant therapeutic relevance. Citation Format: Siva Kumar Natarajan, James Haggerty-Skeans, Joanna Lum, Pranav Narayanan, Stefan Sweha, Sushanth Sunil, Pooja Panwalkar, Jill Bayliss, Peter Sajjakulnukit, Derek Dang, Abhinav Achreja, Deepak Nagrath, Costas Lyssiotis, Sriram Venneti. ZFTA-RELA fusion aberrantly drives glutamine metabolism in lethal pediatric ependymomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6047.
Abstract 833: Exosomes isolation via lyophilization for Philadelphia-positive leukemia therapy
Authors: Fawad Ur Rehman, Rida e Maria Qazi, Zahra Sajid, Afsar Ali Mian
Journal: Cancer Research (2023)

Abstract Background: Exosomes are extracellular vesicles having less than 200 nm size, secreted extracellularly by almost all types of cells. They regulate intercellular communication by delivering RNAi, proteins, viruses, and soluble factors as inherited payloads. In recent years, the application of exosomes to deliver small molecules have been widely investigated in tissue regeneration, therapeutics, as well as diagnostics. However, the isolation of exosomes has remained challenging mainly due to limitations of specialized instruments and the least cost-effective reagents. In this study, we developed a novel method to isolate exosomes from cultured cells. Experimental: The supernatant from cultured cells was collected and subjected to lyophilization after low-speed centrifugation. The characterization and quantification of exosomes were performed using Bradford assay. Scanning Electron Microscopy (SEM) and Nanoparticles Tracking Analysis (NTA) were carried out for shape and size distribution. Western blot was performed for the evaluation of the exosome specific surface markers (CD63, CD9, CD81, calnexin, HSP70, and TSG101). The as-isolated exosomes were loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. Furthermore, their drug loading and release were confirmed by loading tyrosine kinase inhibitors (TKIs) as a payload. Finally, the anti-leukemic drugs (dasatinib and ponatinib) were loaded on the isolated exosomes for targeting leukemia cell line i.e., K562 and BCR-ABL expressing Ba/F3 cells in in vitro model, using MTT assay for cell proliferation and Annexin-V/PI for apoptosis detection. Results: Here we found that the isolated exosomes have round shape with an average size of ~130 nm. They expressed known exosome markers including CD63, CD9, CD81, calnexin, HSP70, and TSG101. These exosomes could efficiently target and deliver dyes (Eosin and Cy5) and TKIs (dasatinib and ponatinib) to K562 and Ba/F3-cells expressing BCR-ABL. The TKIs-loaded exosomes inhibited the growth of leukemic cells and induced apoptosis in a dose dependent manner. Conclusion: We claim the development of a new method for active exosome isolation from cell culture. These exosomes could qualify for all the essential characterization, drug loading, and release ability by harnessing proliferation and inducing apoptosis in Ph+ leukemia cells. Citation Format: Fawad Ur Rehman, Rida e Maria Qazi, Zahra Sajid, Afsar Ali Mian. Exosomes isolation via lyophilization for Philadelphia-positive leukemia therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 833.
Abstract 824: Statistical optimization of stable Quercetin nanoparticles for tumor targeting
Authors: Nourhan Khaled Hassan, Eman El-labbad, Jayalakshmi jagal, Mohamed Haider, Ibrahim El-sayed
Journal: Cancer Research (2023)

Abstract Quercetin is a well-known flavonoid with a significant in vitro anticancer effect and low toxicity profile. Despite of that, its clinical use is still limited because of its low solubility, bioavailability, and instability in physiological conditions. The aim of this study was to develop and optimize stable quercetin nanoparticles targeting tumor cells within the body. Quercetin nanoparticles were synthesized using solvent emulsification technique using zein, beta-cyclodextrin, and pectin at different concentrations. The prepared formulations were optimized using factorial response surface analysis. The stability of the optimized quercetin nanoparticles in conditioned media, obtained from subculture of colorectal cancer cell line HCT-116, was compared to the stability of the free drug powder over 24 h. Moreover, the cytotoxic effect of Quercetin powder and the optimized Quercetin nanoparticles were evaluated against two colorectal cancer lines (HT-29, and HCT116), using MTT assay, under normoxic conditions. Annexin-V/FITC apoptotic assay was used to detect the number of cells that underwent apoptosis using flow cytometry. The optimized formulation was composed of 10 mg of zein and 31.35 mg beta-cyclodextrin, with no pectin. It had particle size of 221 nm, polydispersity index of 0.115, zetapotential of 7.65 mV. The stability of the optimized quercetin nanoparticles in the conditioned media of HCT-116 was significantly higher than the free drug powder over 24 h. In HT-29, the optimized nanoparticles and the free drug had IC50 of 29.33 µM and 60.78 µM, respectively. In HCT-116, optimized nanoparticle and the free drug had IC50’s 31.11 µM and 59.20 µM, respectively. The flow cytometric analysis after annexin-V/FITC staining showed that treatment with the optimized quercetin nanoparticles significantly induced total apoptosis in the two colorectal cancer lines (HT-29 and HCT 116) when compared to the free drug and plain nanoparticles. In conclusion, the optimized quercetin nanoparticles were found to be capable of increasing drug stability in physiological fluids as well as increasing its cytotoxic effect in two colorectal cancer cell lines by apoptosis induction when compared to the drug in powder form as well as plain nanoparticles. Citation Format: Nourhan Khaled Hassan, Eman El-labbad, Jayalakshmi jagal, Mohamed Haider, Ibrahim El-sayed. Statistical optimization of stable Quercetin nanoparticles for tumor targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 824.